7. Project Summary/Abstract Metazoan development, physiology, and evolution are all heavily founded on the activities of transcriptional cis-regulatory modules (CRMs), also known as enhancers. Unraveling the basis of their remarkable regulatory properties is thus a central goal of biological science. While the past decade has been an era of remarkable progress in the study of developmental enhancer modules, we are clearly still in our infancy in understanding exactly how an enhancer's various transcription factor (TF) inputs are properly integrated to generate a novel gene expression output. The research program we propose here is designed to address this fundamental problem directly. Specific Aim 1. Investigate the functional basis for conserved CRM grammar elements. Evolutionarily conserved grammar elements ? pairs of adjacent binding sites for two different TFs ? have emerged as the next frontier in understanding the extraordinary integrative capacity of enhancer modules. We have identified two such elements that mediate synergistic activation by the Achaete/Scute proneural proteins and Suppressor of Hairless, the transducing TF for the Notch signaling pathway. We will conduct a comprehensive series of in vitro and in vivo studies designed to eludicate the biochemical and functional basis of their cooperative action. Specific Aim 2. Elucidate the mechanistic basis of complex CRM activity patterns. Our previous studies have revealed the existence in the Drosophila genome of a large number of enhancer modules that drive expression patterns of surprising spatial and temporal complexity. This discovery raises several intriguing questions. How specifically are these complex patterns generated? Is there a logic that ties together the different components of such a pattern? Do the different subpatterns rely for their generation on distinct subelements of the enhancer? We will carry out detailed structure/function studies of selected sets of complex CRMs, with the goal of testing different models for how they synthesize their output patterns. Specific Aim 3. Investigate the nature and function of a novel conserved CRM in the Enhancer of split gene complex. We have recently identified a novel and deeply conserved enhancer module in the Enhancer of split complex [E(spl)-C]. It lies in an unusual location, and drives an exceptionally broad pattern of reporter gene expression that is unlike that of any other CRM in the Complex. The experiments proposed for Specific Aim 3 will investigate this module's function in detail, including the intriguing possibility that it serves more than one gene, and thus may help explain the long-term evolutionary stability of the E(spl)-C. Transcriptional regulatory sequences are critically important contributors to a broad spectrum of human disease states. By delving deeply into the mechanistic basis of enhancer specificity, our work will help illuminate how regulatory sequence variation affects enhancer output and hence human phenotype.